1. Field of the Invention
This invention relates to urea assay methodology and, more particularly, this invention relates to a colorimetric urea determination method, reagent and reagent kit useful in end point and kinetic urea determination methods.
2. Description of the Prior Art
Various methods of urea demonstration and determination utilize a reaction between urea, o-phthalaldehyde and a chromogenic compound, in which an intensely colored reaction product is produced. For example, Gindler U.S. Pat. No. 4,273,556, the disclosure of which is hereby incorporated by reference, describes a method of urea demonstration and determination in which urea reacts with o-phthalaldehyde and chromotropic acid or one of its salts.
According to the prior Gindler patent, o-phthalaldehyde reacts in an acidic medium with urea present in a liquid sample to form a substantially colorless isoindoline derivative, which in turn reacts with chromotropic acid, or one of its salts, to produce an intensely colored substance of unknown structure whose concentration is linearly related to urea concentration.
Prior o-phthalaldehyde based urea demonstration and determination systems using chromogenic compounds other than chromotropic acid are disclosed in Jung U.S. Pat. No. 3,890,099 and Denney U.S. Pat. Nos. 4,074,972, 4,105,408, 4,131,425 4,131,429 and 4,131,430. The disclosures of each of the foregoing Jung and Denney patents are hereby incorporated by reference.
It has been found in practice that, while the reaction systems described in the prior Gindler, Jung and Denney patents exhibit excellent precision in an end point urea determination, the precision of the kinetic method of analysis, in which absorbance readings of the reaction vessel and a calibrator are taken at two time points and the difference between the absorbances compared, is inferior to the precision of the end point method.
Further, when an aqueous calibrator is used in serum assays, an empirical conversion factor between serum absorbance and serum urea concentrations is required.